C/EBPα has impaired DNA-binding capacity in patients with AML. (A) DNA-binding experiment using a C/EBPα-specific probe with nuclear extracts of 25 000 sorted CD34⁺ AML cells. Nuclear extracts of 25 000 HL-60, 2 independent batches of CB CD34⁺ cells, and 1 batch of CD34⁺ cells isolated from the mobilized peripheral blood of an allogeneic donor are shown as control. Nuclear extracts taken from patient nos. 1 to 11 were mixed, and were used in cold competition experiments using 50-fold excess of unlabeled probe. Supershift experiments were performed with a C/EBPα-specific antibody as well as with control antibodies against GATA1. Data of a representative example of 3 independent experiments are shown. The intensity of C/EBPα DNA binding is indicated below the lanes. (B) EMSA experiment as in panel A, but now nuclear extracts were incubated with a NF-κB–specific probe. Cold competition experiments were performed using 50-fold excess of unlabeled probe.